This application addresses broad Challenge Area (14) Stem Cells and specific Challenge Topic, 14-DE-103: Enhancing Human Embryonic Stem (ES) Cell Culture Systems. The overall objective of this proposal is to develop and optimize a feeder-free in vitro culture system with a controlled culture microenvironment for human pluripotent stem cell growth and directed differentiation. In particular, high-throughput approaches will be used to develop culture materials with optimized surface properties incorporating defined extracellular matrix (ECM) components that allow for accurate control of oxygen partial pressure of the cell surface (pO2cell) to provide more physiological conditions and allow strategies to better mimic normal development processes. The developing embryo is exposed in vivo to very low oxygen levels, and there is growing evidence that oxygen concentration, together with cell-protein interactions, are important factors in differentiation and proliferation. In typical culture systems, pO2cell is unknown because of gradients in the culture medium. Currently, there are no commercially available methods or equipment for culturing cells under known, hypoxic conditions. Membranes of silicone rubber, which have very high oxygen permeability, will be used as a culture substrate so that cells are exposed to the same oxygen level as in the gas phase. Silicone rubber surfaces will be investigated in various forms: (1) unmodified, (2) chemically modified and functionalized with synthetic polymers, and (3) chemically modified with synthetic polymers to which ECM components are attached by physical adsorption or covalent linkage, including defined proteins and mixtures thereof. Cell compatible, ECM protein microarrays on functionalized silicone rubber will be created with a high-throughput microarray platform that we previously developed for culture of cells in order to screen a large number of cell-ECM-biomaterial interactions. The various cell substrates will be examined for cellular attachment, proliferation, and gene expression patterns at various levels of pO2cell using combinatorial techniques. Confirmatory experiments in macro-scale culture using culture vessels will be carried out with the most promising combinations. Similar experiments with the high-throughput microarray platform and culture vessels will be carried out for differentiation of human pluripotent stem cells to cardiomyocytes, which has been shown to benefit from hypoxic culture, as a model system. Accomplishment of the goal of the proposed studies will lead to elimination of conventional feeder layers and undefined xenogenic proteins and to the development and validation of a more well-defined and physiological culture platform for simultaneous variation and study of soluble factors, ECM-cell interactions, and pO2cell, thereby enabling enhancement of our understanding of the role of these factors in maintenance and differentiation of human pluripotent stem cells. This novel, generally applicable platform, will upgrade the capability and quantity of research in this field. Pluripotent stem cells hold enormous promise for drug screening, in vitro modeling of genetic disorders, and cell therapies and the proposed research will have wide impact on human health. At its most basic level, this research will provide information about the interaction of biomaterials, ECM components, and undifferentiated or differentiating human pluripotent stem cells at different known values of pO2cell that mimic physiological conditions, data for which has not previously been available except under normoxic culture. This information will advance our knowledge in the fields of developmental and stem cell biology as well as human pluripotent stem cell technology. In particular, we expect to learn how the choice of ECM proteins and pO2cell levels interact to aid directed differentiation. From a broader standpoint, the proposed high-throughput platform and macro-scale culture vessels that derive therefrom are tools that constitute enabling technology to culture human pluripotent stem cells under defined physiological conditions. They will improve the research infrastructure in terms of the capability for culturing human pluripotent stem cells. The knowledge acquired and tools developed may have profound effects on a wide variety of applications in many fields of human health. This will accelerate developments in applications such as drug screening, in vitro models of genetic disease, therapeutic replacement of diseased cells in major diseases such as heart disease, diabetes, and neural diseases such as Parkinson's, which annually affect millions of people in the United States. Indeed, differentiation of human pluripotent stem cells to cardiomyocytes, the proposed model system, is itself of high interest for generating cells and tissues for repair of cardiac tissues following ischemic heart disease. Furthermore, the culture vessels to be tested will be useful in culturing cardiac tissue. This collaborative study will integrate recent progress in the fields of bioengineering, biomaterials, and developmental biology to design a new culture platform and protocols that will facilitate cell-based therapies for treatment of a multitude of human diseases and other ES cell applications. PUBLIC HEALTH RELEVANCE: This project concerns improved methods to work with human stem cells so that they can develop into medically useful cells and tissues. For example, this can lead to ways to grow functioning heart muscle cells that can be used to treat heart disease.